A common method used to separate analytes within a sample is liquid chromatography. Liquid chromatography employs specific chromatographic columns and one or more mobile phases used to both equilibrate the column and elute analytes therefrom. Chromatography columns are used to effectuate the separation, purification and study of analytes contained within a homogeneous or heterogeneous sample. Columns are packed with sorbent material (also referred to as the “stationary phase” or “packing bed”) that provides a chemical milieu with which analytes of a sample can interact. Generally, the sorbent material contains functional groups having a specific chemistry. For example, reverse-phase columns have a stationary phase comprising molecules with one or more hydrophobic groups. These hydrophobic groups, e.g., a C18 hydrocarbon chain, will interact with other molecules via hydrophobic interaction. This hydrophobic interaction can be interrupted with an organic mobile phase, thus eluting analytes from the stationary phase.
The most commonly used chromatographic columns, referred to herein as “conventional columns”, are comprised of a column tube into which sorbent is packed, and inlet and outlet end fittings, which facilitate connection to the fluid stream, and which also contain filters that are designed to contain the sorbent bed within the column tube itself. These filters are disposed at each end face of the column tube.
In operation, the sorbent bed within the chromatographic column is subjected to fluid forces resulting from the flow of mobile phase through the column. Over time, these forces may disrupt the packing bed of the column resulting in voids or dead volume (V0) within the column's packing bed. These voids have a detrimental effect on the performance of a column. Voids can in effect serve as mixing chambers within the confines of the column leading to the loss of column efficiency. For example, the practitioner may observe excessive peak tailing over time using a standard analyte, such peak tailing is indicative of a poor column.
An issue that plagues practitioners of chromatography is how to mitigate this loss of column performance. Some chromatography columns in the prior art employ a movable device situated within the column. This movable device, for example a piston, is designed to adjust the internal volume of the chromatographic column in response to changes in the sorbent bed density during use, thereby attempting to minimize the void volume within the column. However, these designs are typically very complex requiring multiple components and seals and are therefore inherently more expensive to produce. Additionally, the columns with these movable devices require that the column tube itself be significantly larger and therefore much less convenient to use. Further, these columns with movable devices do not contain a fixed volume of sorbent, which can lead to difficulty in predicting analyte retention times and the scaling of chromatographic methods across different column sizes
Thus, there currently exists a need for a chromatography column that minimizes the effects of sorbent bed compression, which incorporates a simple, cost effective design, is highly portable and convenient to use, and does not negatively affect the column's chromatographic behavior. The present invention addresses this need in toto. In addition, the present invention provides for a method of increasing the sorbent bed density within a chromatographic column beyond what is possible with conventional fixed length column designs, thereby improving the stability of the column during use.